Camel Milk Based Pharmaceutical Composition

ABSTRACT

The present invention relates to a novel pharmaceutical composition with milk of immunized camelid, particularly  Camelus dromedaries  as the main component (i.e. active ingredient) and a method for the treatment or prevention of skin or mucus membrane infections, particularly Acne vulgaris. The present invention also discloses a process of preparing said composition.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a continuation of U.S. application Ser. No.13/609,342 filed Sep. 11, 2012, titled “Camel Milk Based PharmaceuticalComposition.”

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition for thetreatment or prevention of skin or mucus membrane infections such asacne, and more particularly, the invention relates to a pharmaceuticalcomposition based on immunized camelid milk, particularly Camelusdromedarius as the active ingredient, and a process of preparing thecomposition.

BACKGROUND OF THE INVENTION

Acne vulgaris caused by the bacterium Propionibacterium acne (P. acne)is the most common cutaneous disorder with a prevalence of 70-85% inadolescents. Although acne is not a life-threatening disease, it hassignificant physical and psychological effects such as permanentscarring, poor self-image, social inhibition, depression, anxiety, andsuicidal tendency. Therefore, acne may be regarded as a serious medicalcondition. Topical therapy is inevitable in acne treatment and is mainlyindicated in the mild to moderate acne. In more severe forms, a combinedtopical and systemic therapy is recommended. The available topicalagents have a direct or indirect influence on the patho-genetic factorsand are selected according to the predominant type of acne lesions. Thetherapeutic success in acne and related skin disorders are highlydependent on a regular application of the topical agents over aprolonged period of time. However, disadvantages associated with thecommonly used topical agents considerably affect the patient complianceand obstruct the treatment. Currently, available treatment for acne andrelated skin disorders is mostly based on antibiotics and retinoids. Theuses of antibiotics have a lot of limitations due to development ofresistance by bacteria. On the other hand, Retinoids are highlyteratogenic.

Animal milk has been used in the preparation of pharmaceutical andcosmetic compositions. Milk of ruminants, and predominantly bovine milk,has been utilized most. Among drawbacks of cow milk is a wide-spreadallergy to it, affecting in several of its forms as much as 50%individuals in some populations. It is an object of this invention toprovide a composition comprising immunized milk which keeps all thebenign properties of milk but is free of the drawbacks related to cowmilk. Camel milk has been traditionally used by certain ethnic groups,and it was found that, in some respects, its composition is closer tothe human milk than cow milk.

Passive immunity is provided to newborns by Immunoglobulins present incolostrum until its own immune system matures. The concentration incolostrum of specific antibodies against pathogens can be raised byimmunizing a mammal with these pathogens or their antigens. Immunizedmilk products are preparations made of such hyper-immune colostrum orantibodies enriched from it. These preparations can be used to giveeffective specific protection against different diseases. Colostralimmunoglobulin supplements designed for farm animals are commerciallyavailable in many countries. Also, some immunized milk products thatcontain specific antibodies against certain pathogens have been launchedin the market. A number of clinical studies are currently in progress toevaluate the efficacy of immunized milks in the prevention and treatmentof various human infections, including those caused by antibioticresistant bacteria. Bovine colostrum-based immunized milk products areused as prophylaxis against various infectious diseases in humans.Immunized milk products are examples of health-promoting functionalfoods, or nutraceuticals.

SUMMARY OF THE INVENTION

It is an object of the present invention to eliminate the disadvantagesof the prior art by providing a pharmaceutical composition comprisingtreated immunized camelid milk in a pharmaceutically acceptable vehiclefor the treatment or prevention of skin or mucus membrane infections.

A second object of the present invention, is to provide a process ofpreparing the composition comprising;

-   -   a. Immunizing a female camelid with a vaccine of        propionobacterium acne, Staphylococcus aureus, Streptococcus        mutans, Pseudomonas aeroginosa, Haemophilus influenza,        Neisseriae spp. Candida spps or Chlamydia trachomatus;    -   b. Obtaining milk from the camelid;    -   c. Treating the milk at a temperature of 0° C. for 1-2 hours and        centrifuging at 15000 rpm to remove lipids;    -   d. Treating the milk with rennin or acetic acid to reduce        protein content;    -   e. Pasteurizing the milk at a temperature ranging from 65° C. to        72° C. for 15 minutes; and    -   f. Preparing the milk in a pharmaceutically acceptable vehicle.

Another object of the present invention is to provide a method for thetreatment or prevention of skin or mucus membrane infections, comprisingapplying to infected areas of the human body an effective amount of thecomposition of the invention.

Preferably, the camelid described in the invention is chosen from thegenus Camelus, Llama, or Vicuna. More preferably, the camelid is Camelusdromedaries.

The camelid described in the invention is immunized with skin or mucusmembrane pathogen, preferably propionobacterium acne, Staphylococcusaureus, Streptococcus mutans, Pseudomonas aeroginosa, Haemophilusinfluenza, Neisseriae spp. Candida spps or Chlamydia trachomatus.

Preferably, the pharmaceutical composition of the invention is in theform of cream, ointment, gel, skin wash, lotion, soap, shampoo, mouthwash, vaginal wash, eye wash, tooth paste, or spray.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows whey of immunized camel tested by Enzyme-LinkedImmunosorbent Assay (ELISA) and the kinetic response over the experimentperiod.

FIG. 2 shows Coomassie SDS-PAGE gel (12.5%) for P. acne camel whey. Therun was done for 30 min at 100V, then for 45 min at 150V. Load was 20μl/well. Dilution was (3:1) with 4× dye for all fractions.

FIG. 3 shows bactericidal activity of whey obtained from milk ofimmunized camel on P. acne.

FIG. 4 shows the growth inhibition of whey obtained from milk ofimmunized camel.

FIG. 5 shows anti-acne activity of aqueous cream extract.

DETAILED DESCRIPTION OF THE INVENTION

The pharmaceutical composition of this invention comprises immunizedcamelid milk as the active ingredient in a pharmaceutically acceptablevehicle, in addition, a method for treatment or prevention of skin ormucus membrane infections is described, for example, acne vulgaris.

The camelid, preferably Arabian camel (Camelus dromadarius), isimmunized subcutaneously with an initial dose of 3 ml of prepared skinor mucus membrane pathogen, for example, Propionibacterium acne vaccine.The vaccinated camel is boosted 4 times at 2 week intervals with 5 ml ofvaccine for each booster.

The Propionibacterium acne vaccine is prepared from P. acnes (NCTC 373),as shown in Examples 1 and 2.

The pharmaceutical composition, according to the present invention, isintended for treating the skin, and may be in a pharmaceuticallyacceptable vehicle in the form which include, but is not limited toliquid, paste, or solid, and more particularly in the form of ointment,cream, milk, powder, impregnated pad, wipes, solution, gel, spray,suspension, lotion, shampoo, or washing base. The composition may alsobe in the form of suspension of lipid or polymer vesicle, nano sphere ormicro sphere, or polymer patches and hydro-gels allowing a controlledrelease. The composition may be in anhydrous form, in aqueous form, orin the form of an emulsion. The components of the pharmaceuticalcomposition and their ratios could be adjusted according to thepharmaceutically acceptable vehicle and the intended application of thepharmaceutical composition. In a preferred embodiment of the presentinvention, the pharmaceutical composition is in the form of a cream, andin another preferred embodiment of the present invention, thepharmaceutical composition is in the form of a gel.

The present invention further relates to a process of preparing thepharmaceutical composition, comprising:

-   -   1. Immunizing a female camel with a vaccine of intact or whole        cell lysate of P. acne;    -   2. Obtaining milk from the female camel;    -   3. treating the female camel milk at a temperature of 0° C. for        1-2 hours and centrifuging at 15000 rpm to remove lipids;    -   4. Treating the milk with rennin or acetic acid to reduce        protein content;    -   5. Pasteurizing the camel milk at a temperature ranging from        65° C. to 72° C. for 15 minutes; and    -   6. Preparing the camel milk in a pharmaceutically acceptable        vehicle.

The invention will be further described and illustrated in the followingexamples.

EXAMPLES

The following examples illustrate the present invention without,however, limiting the same thereto.

Example 1

Cell Culture

P. acnes (NCTC 373) were cultured on Mueller Hinton Agar (MHA) underanaerobic conditions using Gas-Pak (Oxoid) at 37° C. for 72 hrs.Standard inoculums of Optical Density 600 (OD600)=1.0-3.0 were preparedby inoculating colonies of P. acne in Phosphate Buffered Saline (PBS).

Example 2

Bacteria Harvesting

-   -   1. The bacterial cells were harvested by adding 1 ml of        autoclaved PBS 1× to the growing bacteria and collected from the        plate with a sterile Pasteur pipette;    -   2. The bacteria were washed 3 time with PBS;    -   3. The suspension was prepared as 2 McFarland standard/1.5 ml;    -   4. The bacteria were inactivated by heating at 60° C. for 30 min        and centrifuged at 5,000×g for 5 min;    -   5. The bacteria were mixed with PBS until reaching the required        McFarland/1.5 ml; and

1.5 ml of the inactivated bacteria were mixed with 1.5 ml of theFreund's adjuvant complete (SIGMA) and injected subcutaneously into acamel.

Example 3

Camel Immunization

For each infection model, two adult female Arabian camels (Camelusdromedarius) were used; one as control and the other was immunizedsubcutaneously with an initial dose of 3 ml of preparedPropionibacterium acne vaccine. The vaccinated camel was boosted 4 timesat 2 week intervals with 5 ml of the vaccine for each booster. The twocamels were kept in a farm for two months, and were kept under similarconditions.

Example 4

Detection of Specific Antibodies Against Propionibacterium acne in Milkby Enzyme-Linked Immunosorbent Assay (ELISA)

For milk antibody screening, the ELISA was performed. Flat bottomed 96well polystyrene micro titer plates (Greiner, Germany) were coated with100 μl of 10 μg/m Propionibacterium acne antigens incarbonate-bicarbonate buffer (pH 9.6) overnight at 4° C. The plates werewashed 3 times with 100 μl of 0.15 M PBS (pH 7.2) containing 0.05% Tween20 and blocked with 100 μl of 2% Bovine Serum Albumin (BSA) in PBS forone hour at Room Temperature (RT). The plates were washed again and 100μl of the serum samples diluted at 1:100 in 1% BSA were added induplicates and incubated for one hour at RT, whereas, milk samples wereadded in duplicates without dilution. Negative and positive controlsamples were incorporated in each of the plates. After washing, 100 μlof one of the horse radish peroxidase (HRP) conjugated protein A andprotein G were diluted at 1:1000 in 1% BSA, and were added separately toeach well. The plates were incubated for one hour at RT and washedagain. Finally, 100 μl of 0.1% O-phenylenediamine (Sigma, USA)containing hydrogen peroxide in 0.1 M citrate buffer (pH 4.5) were addedto each of the wells and absorbance was measured at 490 nm using ELISAreader (AsysHitech, Switzerland). Results are shown in FIG. 1.

Example 5

Identification of Camel Milk Proteins Using Sodium Dodecyl SulfatePolyacrylamide Gel Electrophoreses (SDS-PAGE)

Components of camel skimmed milk were fractionated by SDS-PAGE (afterprecipitation of casein). This was performed using Desaphor VE minigel(Heidelberg, Germany) in the discontinuous buffer system using 0.5 mmthick 10% acrylamide-bisacrylamide gels under non-reducing conditions asdescribed by Hamers-Casterman et al. (1993). For preparing resolvinggels, 4 mL distilled water, 2.5 mL running buffer (pH 8.8), 3.3 mL of30% acrylamide-bisacrylamide solution, 100 μL of 10% ammonium persulfate(APS) and 10 μL Tetramethylethylenediamine (TEMED) were mixed. Stackinggels (4%) were prepared by adding 6 ml of distilled water, 2.5 mlstaking buffer (pH 6.6), 1.3 mL of 30% acrylamide-bisacrylamidesolution, 100 μl of 10% Ammonium persulfate (APS), and 10 μl TEMED. Themilk was mixed with an equal volume of sample buffer lackingB-mercaptoethanol (non-reducing conditions) (pH 6.8). For band sizedetermination, molecular weight protein standard was used after beingprocessed in a similar way as the milk samples. Electrophoresls wascarried out using running buffer with pH 8.3 at 120 volts for 60-120minutes. The gel was stained with Coomassie brilliant blue R-250 (exceptif used for immunoblot) and destained by 20% acetic acid until clearbands were seen. Results are shown in FIG. 2.

Example 6

In Vitro Activity of Whey Against Acne

Antibodies isolated from whey of Camel milk immunized with killed P.acne are mixed with a standard inoculum of the bacterium andsubsequently incubated for 72 hrs. The total count is determined andthen compared with the total count of the standard inoculum withoutmixing with the antibodies. Results are shown in FIG. 3 and FIG. 4.

Example 7

Preparation of Cream Formula

For the treatment of acne, a cream formula has been prepared from thefollowing components:

Cream Formula 0.5% w/w Material weight mg/unit Camel dried whey* 15 g2.5 Cetyl alcohol 90 g 15 Tween 20 30.9 ml 5.15 Glycerylmonostearatetype1 17.1 g 2.85 MERKUR 791 60 g 10 Purified water 387 ml 64.5 Total600 100 *Equivalent to 0.5% w/w total protein

Preparation Protocol:

-   -   1. Cetyl alcohol, Glyceryl monostearate and Merkur were molten        in same container at 75° C. using water bath;    -   2. Tween 20 was mixed with purified water;    -   3. Camel whey powder was dissolved in Tween mixture;    -   4. The mixture from step 3 was heated to 45°-50° C.;    -   5. The mixtures from step 1 was cooled to 65°-70° C.; and    -   6. Both mixtures were mixed together and cooled while mixing        until cream turns white.

Example 8

Preparation of Gel Formula

For the treatment of acne, a gel formula has been prepared from thefollowing components:

Gel formula 0.5% w/w Material weight mg/unit Camel dried whey* 8.75 g2.5 Hydroxypropyl cellulose grade HF 10.5 g 3 Deionized (D.I.) water330.75 ml 94.5 Total 350 g 100 *contains 0.5% total protein.

Preparation Protocol:

-   -   1. Camel dry whey was dissolved in D.I. water;    -   2. Hydroxypropyl cellulose grade HF was dissolved in solution        from step 1;    -   3. The mixture was continuously mixed until a clear homogenous        gel has formed; and    -   4. The gel was filled in tubes.

Example 9

Recovery of Active Ingredients from Cream

The active ingredients of camel milk whey were re-extracted from thecream for the purpose of stability testing. The cream was mixed withequal volumes of PBS and incubated in water bath at 56° C. for 10minutes. The mixture was then centrifuged at 13400 rpm for 10 minutes,using microfuge. The aqueous phase was collected and tested using ELISA.Results are shown in FIG. 5.

Example 10

In Vivo Testing of the Efficacy of Whey Protein Concentrate of Milk ofImmunized Camel Against Propionibacterium acne

Protocol:

Propionibacterium acne (10⁸ CFU/ml (OD₆₀₀=2)) bacteria were mixed withthe whey protein concentrate and incubated for 2 hrs before Intradermalinjection in the central portion of rabbit ear. Both positive whey andnegative whey were used and compared. Injection was gradually performedby 28 gauge needle to prevent leakage. Gross examination was performeddaily along the period of investigation (two weeks). Histopathologicalevaluation was performed after sacrificing the animals.

Results:

Gross Examination:

Ear thickness: the infection induced rabbit showed double fold increasedear thickness compared to the normal non infected ear. In contrast, earthickness was significantly reduced in cases of injecting pretreatedbacteria with positive whey and less significantly in cases of negativewhey. In addition, no significant differences were observed concerningredness, heat, formation of papules and pus.

Histopathological Examination:

In the infection induced rabbit, the inflammatory response was presentedwith dense mixed inflammatory cells and marked eosinophilia, formationof micro abscess and ulcerations without affecting the epidermis. Incontrast, mild mixed inflammatory cells without ulceration and lessmicro abscesses were markedly observed in cases of injecting pretreatedbacteria with positive whey. No differences were observed in thenegative whey treated rabbit in comparison to the infection induced ear.

Microbiological Examination

The recovery of viable P. acne was possible in cases of pus formation.No differences were seen among all groups.

While the present invention has been described in details and withreference to specific embodiments thereof, it will be apparent to oneskilled in the art that various additions, omissions and modificationscan be made without departing from the spirit and scope thereof.

1. A pharmaceutical composition comprising treated immunized Camelidmilk in a pharmaceutically acceptable vehicle for the treatment orprevention of skin or mucus membrane diseases.
 2. The pharmaceuticalcomposition of claim 1, wherein said Camelid is from the genus Camelus,Llama, or Vicuna.
 3. The pharmaceutical composition of claim 2, whereinsaid camelid is Camelus dromedarius.
 4. The pharmaceutical compositionof claim 1, wherein said camelid is immunized with skin or mucusmembrane pathogen.
 5. The pharmaceutical composition of claim 4, whereinsaid pathogen is Propionobacterium acne, Staphylococcus aureus,Streptococcus mutans, Pseudomonas aeroginosa, Haemophilus influenza,Neisseria OJ spp. Candida spps or Chlamydia trachomatus.
 6. Thepharmaceutical composition of claim 5, wherein said pathogen is intactor any a fraction treated with heat, formalin or cell lysate.
 7. Thepharmaceutical composition of claim 1, wherein said immunized camel milkis treated at a temperature of 0° C. for 1-2 hours and centrifuged at15000 rpm for the removal of lipids.
 8. The pharmaceutical compositionof claim 1, wherein said immunized camelid milk is treated enzymaticallyor chemically to reduce protein content.
 9. The pharmaceuticalcomposition of claim 8, wherein said enzyme is rennin, and said chemicalis acetic acid.
 10. The pharmaceutical composition of claim 1, whereinsaid composition can be in the form of cream, ointment, gel, skin wash,lotion, soap, shampoo, mouth wash, vaginal wash, eye wash, tooth paste,or spray.
 11. A process of preparing the pharmaceutical composition ofclaim 1, comprising; Immunizing a female camelid with a vaccine ofPropionobacterium acne, Staphylococcus aureus, Streptococcus mutans,Pseudomonas aeroginosa, Haemophilus influenza, Neissenriae spp. Cancfidaspps or Chlamydia trachomatus; Obtaining milk from the camelid; Treatingthe milk at a temperature of 0 nc for 1-2 hours and centrifuging at15000 rpm to remove lipids; Treating the milk with rennin or acetic acidto reduce protein content; Pasteurizing the milk at a temperatureranging from 65° C. to 72° C. for 15 mmutes; and Preparing the milk in apharmaceutically acceptable vehicle.
 12. The process of claim 11,wherein said camelid is from the genus Camelus, Llama, or Vicuna. 13.The process of claim 12, wherein said camelid is Camelus dromedarius.14. The process of claim 11, wherein said composition can be in the formof cream, ointment, gel, skin wash, lotion, soap, shampoo, mouth wash,vaginal wash, eye wash, tooth paste or spray.
 15. A method for thetreatment or prevention of skin or mucus membrane infections, comprisingapplying to an infected area of the human body an effective amount ofthe composition of claim
 1. 16. The method of claim 15, wherein saidinfections are caused by Propionobacterium acne, Staphylococcus aureus,Streptococcus mutans, Pseudomonas aeroginosa, Haemophilus influenza,Neisseriae spp. Candida spps or Chlamydia trachomatus.